Recording intracellular signals is a key source of data in the fields of genomics, cell biology and neuroscience. For example, in genomics and cell biology, patterns of gene expression and protein interactions are measured by monitoring concentrations of key molecules within the cell. In neuroscience, recordings of voltage differences across a cell membrane or of concentrations of free ions provide investigators access to the temporal pattern of intracellular action potentials (one millisecond, 100 millivolt (mV) pulses) with which nearly all computation in the brain is performed.
Biological organisms are composed of various structures, which are composed of large numbers of cells of a wide variety of types. For example, the nervous system is composed of billions of cells, i.e., neurons, that communicate using patterns of action potentials relayed across anatomically specified connections. Traditional methods for recording from a neuron involve accessing the intracellular space of the neuron at a point where it is on the order of about ten to about 25 micrometers in diameter, with a glass microelectrode, amplifying the voltage difference between the inside and outside of the cell and recording the amplified signal using a recording device. For a description of a method involving accessing the intracellular space of a neuron, see A. L. Hodgkin et al., Measurement of Current-Voltage Relations in the Membrane of the Giant Axon of Loligo, J. PHYSIOL., 116, 424-448 (1952). Accessing multiple neurons requires the use of multiple electrodes, each connected to a separate amplifier and recording device. The mechanical constraints on recording from such small biological structures have limited the proximity and number of recordings that can be made simultaneously. Even though advances have expanded capabilities to allow for the recording of larger numbers of neurons, constraints still exist. Current techniques are limited to recording up to only one hundred neurons simultaneously. See J. D. Kralik et al., Techniques for Long-Term Multisite Neuronal Ensemble Recordings in Behaving Animals, 25 METHODS, 121-51 (2001).
To fully characterize a biological structure, recordings from a large number of cells are required. These recordings need to be analyzed off-line to determine temporal correlations, cause and effect interactions and potential communication and information processing strategies implemented by the structure. Due to the limitations on the number of simultaneous recordings currently possible, recordings from different experiments, performed at different times, and on different specimens, are combined to conduct these off-line analyses. Combining results from different experiments can often obscure the underlying dynamics of any one of the experimental preparations studied and therefore lead to incorrect conclusions, e.g., regarding the overall function of a structure.
An alternate approach for intracellular recording involves the use of optical methods in which intracellular signals are translated into fluorescence signals via an artificially introduced intracellular fluorophore. See D. Smetters et al., Detecting Action potentials in Neuronal Populations with Calcium Imaging, 18 METHODS 215-221. These optical methods solve the problem of mechanically accessing the intracellular spaces of multiple cells simultaneously since the light emitted from cells under fluorescent illumination is recorded using microscopic video recordings. However, most biological tissue is highly light-scattering, so resolution of single cells becomes impossible as the focal plane advances to only one millimeter (mm) inside a structure, even when the most advanced microscopic techniques are employed to collect the emitted light. Furthermore, the field of view and numerical aperture of the light collecting apparatus limits the size of a structure that can be imaged, and thus the number of cells that can be recorded simultaneously using these methods. Chimeric fluorescent proteins have been synthesized that monitor cellular signals. However, they do not overcome the shortcomings highlighted above.
Another alternative approach for recording signals from cells involves the use of functional magnetic resonance imaging (fMRI) or positron emission tomography (PET). Both methods can record from very large regions of brain tissue. The spatial resolution of these methods is limited to millimeter-scale, however, and the temporal resolution to hundreds of milliseconds. Therefore, intracellular recordings of action potentials using either of these methods are theoretically impossible, given the much smaller size of single neurons, e.g., less than 0.05 mm, and much briefer time-scale of the action potential, e.g., about one millisecond. Instead, what fMRI and PET imaging provide is access to an average signal from hundreds of thousands of adjacent neurons. While these recordings can be highly informative about brain function, they cannot decompose brain structures to the level of the single neuron, and thus cannot analyze neural computation within these structures.
It would be desirable to have a technique for cellular analysis that does not suffer from the above and other limitations, such that comprehensive study of biological structures and processes may be realized.